Team:Heidelberg/Project dual assay plasmid

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Revision as of 00:02, 22 October 2009

Dual Assay Plasmid

Introduction

In order to control transfection efficiencies, we co-transfected pSMB_MEASURE (which expresses GFP under the control of the promoter of interest) with pSMB_REFERENCE (which expresses mcherry under the control of the JeT constitutive promoter). In flow cytometrie, we were able to first gate for the red fluorscence of mCerry and then for the GFP, which allowed for proof of transfection efficiencies. A more advanced method would bes to have both promoters and reportergenes on one plasmid. That way a 1:1 ratio of reference and measured part could be achieved, providing a standardized comparison of promoter strength, due to the elimination of different transfection efficiencies. This new way of measurement could be achieved by the following plasmid (Fig. 1)which we tried to clone during the summer.

Results

Unfortunately the repeated cloning of the construct was not successful (see Dual assay plasmid).

Discussion

Although we had several cloning strategies and tried each of them many times we were not able to build this advanced measurement plasmid. One probable reason for the failure of that subproject could be the size of the plasmid: The inserte became very large and without advanced cloning tools, we were not able to ligate them into the backbone.

Figure 1: The planned dual assay plasmid contained a site for promoter exchange, where one can put the promoter, that has to be tested. This promoter is upstream of a GFP gene and regulates its transcription. This gene is followed by a Jet promoter that constitutively promotes transcription of a mCherry, serving as a reference.

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 === Introduction ===
-In order to control transfection efficiencies, we co-transfected pSMB_MEASURE (which expresses GFP under the  control of the promoter of interest) with pSMB_REFERENCE (which expresses mcherry under the control of the JeT constitutive promoter).  It would be advantageous to have both promoters and fluorescent proteins on one plasmid to achieve a 1:1 ratio of reference and measured part. This construction would allow a standardized comparison of promoter strength, due to the elimination of different transfection efficiencies. Unfortunately the repeated cloning of the construct, which is shown in Fig. 1, was not successful. Probably the plasmid was becoming too large to be assembled without advanced cloning tools.
+In order to control transfection efficiencies, we co-transfected pSMB_MEASURE (which expresses GFP under the  control of the promoter of interest) with pSMB_REFERENCE (which expresses mcherry under the control of the JeT constitutive promoter). In flow cytometrie, we were able to first gate for the red fluorscence of mCerry and then for the GFP, which allowed for  proof of transfection efficiencies. A more advanced method would bes to have both promoters and reportergenes on one plasmid. That way a 1:1 ratio of reference and measured part could be achieved, providing a standardized comparison of promoter strength, due to the elimination of different transfection efficiencies. This new way of measurement could be achieved by the following plasmid (Fig. 1)which we tried to clone during the summer.
+=== Results===
+Unfortunately the repeated cloning of the construct was not successful (see[[Team:Heidelberg/Notebook_Measurement#Dual assay plasmid| Dual assay plasmid]]).
+=== Discussion===
+Although we had several cloning strategies and tried each of them many times we were not able to build this advanced measurement plasmid. One probable reason for the failure of that subproject could be the size of the plasmid: The inserte became very large and without advanced cloning tools, we were not able to ligate them into the backbone.
 [[Image:Dual_plasmid.png|thumb|left|300px|<div style="text-align:justify;">'''Figure 1: The planned dual assay plasmid contained a site for promoter exchange, where one can put the promoter, that has to be tested. This promoter is upstream of a GFP gene and regulates its transcription. This gene is followed by a Jet promoter that constitutively promotes transcription of a mCherry, serving as a reference.</div>]]